Placental insufficiency disrupts cardiomyocyte ploidy and cell cycle fate in growth-restricted fetal sheep.

Abstract

Cardiomyocytes undergo proliferation, differentiation, and hypertrophy during fetal development. Current techniques struggle to distinguish cardiomyocyte proliferation from alternative cell fates. In this study, we combined flow cytometry measures of cardiomyocyte ploidy (DAPI) and in vivo DNA replication (EdU) over a 24-h period to evaluate the trajectories of cardiomyocytes from normally growing control fetal sheep and fetuses affected by placental insufficiency and fetal growth restriction (FGR) at 0.9 gestation. We categorized ∼100,000 cardiomyocytes from the left and right ventricles (LV and RV) of each animal as proliferating (2C EdU), differentiated (4C EdU), or endoreplicating and polyploid (6C+ EdU). Compared with controls, FGR hearts had 25%-50% fewer cardiomyocytes that replicated DNA (EdU) (LV:= 0.02, RV:= 0.002). The fraction of proliferating cardiomyocytes, indicated by the population of newly synthesized 2C EdUdaughter cells, was ∼20% lower in FGR fetuses (LV:= 0.006, RV:= 0.02). Instead, the percentage of endoreplicating cardiomyocytes (6C+ EdU) in FGR hearts was double that of controls (LV:= 0.004, RV:= 0.002). Although total EdUwas not a strong predictor of cardiac growth, LV and RV mass correlated positively with the percentage of 2C EdUcardiomyocytes and negatively with 6C+ EdUcardiomyocytes across all fetuses. LV mass also correlated positively with the percentage of differentiated cardiomyocytes (4C EdU), which was lower in FGR hearts compared with controls (= 0.008). This study is the first to characterize cardiomyocyte fate following DNA replication in fetal sheep. Our findings suggest that FGR cardiomyocytes differentially prioritize their cycling capacity in favor of polyploidization instead of proliferation.Knowledge of cardiac development has been limited by available methodologies. We used a novel flow cytometry approach to measure DNA replication in utero and distinguish between cardiomyocyte proliferation, differentiation, and endoreplication in growth-restricted (FGR) and normally growing fetal sheep. FGR cardiomyocytes have lower proliferation rates but increased endoreplication compared with controls. Endoreplication and polyploidy are negatively correlated with ventricular mass. Our findings provide insight into fetal cardiac development and how cardiomyocyte fate is altered by FGR.